Utetheisa Ornatrix) (Nuptial Gift͞sexual Selection͞pyrrolizidine Alkaloids͞lepidoptera͞arctiidae)
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Proc. Natl. Acad. Sci. USA Vol. 96, pp. 5570–5574, May 1999 Ecology Sexually transmitted chemical defense in a moth (Utetheisa ornatrix) (nuptial giftysexual selectionypyrrolizidine alkaloidsyLepidopterayArctiidae) ANDRE´S GONZA´LEZ,CARMEN ROSSINI,MARIA EISNER, AND THOMAS EISNER* Section of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853 Contributed by Thomas Eisner, March 17, 1999 ABSTRACT The arctiid moth Utetheisa ornatrix is pro- the females with protection. The tests were coupled with a tected against predation by pyrrolizidine alkaloids (PA) that quantitative assessment of the allocation of the male’s PA gift it sequesters as a larva from its food plant. Earlier work had to different parts of the female’s body and with a determina- shown that males transmit PA to the female with the sperm tion of the deterrence of PA to one of the spiders (L. package and that the female bestows part of this gift on the ceratiola).† eggs, protecting these against predation as a result. We now show that the female herself derives protection from the gift. MATERIALS AND METHODS Females deficient in PA are vulnerable to predation from spiders (Lycosa ceratiola and Nephila clavipes). If mated with a Utetheisa. Several categories of adult Utetheisa were offered PA-laden male, the females become unacceptable as prey. The in the tests with spiders. effect takes hold promptly and endures; females are unac- Field-collected (wild) Utetheisa. These were netted as adults ceptable to spiders virtually from the moment they uncouple in and around patches of Crotalaria mucronata, on the grounds from the male and remain unacceptable as they age. Chemical of the Archbold Biological Station, near Lake Placid, High- data showed that the female allocates the received PA quickly lands County, FL. Such moths contain usaramine (1), the to all body parts. We predict that other instances will be found principal PA in C. mucronata (8). The moths were kept in of female insects being rendered invulnerable by receipt of individual vials with access to water (soaked cotton wad) and sexually transmitted chemicals. offered to N. clavipes within 2 days of capture. Laboratory-reared (2) Utetheisa and (1) Utetheisa. For years, we have raised Utetheisa in our Cornell laboratories on The moth Utetheisa ornatrix (Lepidoptera: Arctiidae; hence- two types of larval diet: one with and one without PA. Details forth referred to as Utetheisa) depends on pyrrolizidine alka- of the composition of these diets are given elsewhere (9). loids (PA) for defense. It sequesters the PA as a larva from its Briefly, the PA-free diet [(2) diet] is based on pinto beans, food plants (Fabaceae; Crotalaria spp.) and retains the chem- which are free of PA, whereas the PA-containing diet [(1) icals through pupation into adulthood. Females also transmit diet] includes a supplement of seeds of Crotalaria spectabilis, PA to their eggs (1). All developmental stages of the moth are another major food plant of Utetheisa. Utetheisa raised on these protected as a result: the larvae and adults against spiders (2, diets are designated (2) Utetheisa and (1) Utetheisa, respec- 3) and the eggs against ants and ladybird beetles (4, 5). tively. The (1) Utetheisa had been shown to contain mono- Notably, the PA in the eggs do not stem entirely from the crotaline (2), the principal PA in C. spectabilis (3, 10). mother moth. Mating in Utetheisa involves transfer from male to female of a sperm package (spermatophore) of considerable size, amounting on average to over 10% of male body mass (6). In addition to sperm and a quantity of nutrient that the female invests in egg production (7), the spermatophore contains PA. It has been shown experimentally that in endowing the eggs, the female resorts not only to her own PA but also to a portion of the PA received from the male (4). The question of whether the female herself might benefit from receipt of the male’s alkaloidal gift remained unan- swered. Might she derive a defensive advantage from mating? Here, we provide evidence that she does and that the advan- tage takes hold virtually from the moment the female uncou- ples from her partner. Our experiments consisted of testing for the vulnerability of adult Utetheisa to predation by orb-weaving spiders (field tests Once-mated laboratory-raised females [2 / (1 ?) and 2 / with Nephila clavipes; Araneidae) and wolf spiders (laboratory (2 ?)]. These females were raised on the (2) diet and were tests with Lycosa ceratiola; Lycosidae). The Utetheisa offered mated either with a (2) male and designated 2 / (2 ?)or to the spiders were of several categories, including, most witha(1) male and designated 2 / (1 ?). Matings were importantly, females that had been raised to be PA-free but effected by confining 2-day-old virgin males and females, in that had mated with males that were either PA-laden or PA-free. These categories permitted us to determine whether individual pairs, in small chambers (8-cm diameter and height) the alkaloidal gift from the male is itself sufficient to provide overnight. Pairs typically remain in copula for over 9 h (11). The publication costs of this article were defrayed in part by page charge Abbreviation: PA, pyrrolizidine alkaloid(s). *To whom reprint requests should be addressed. e-mail: payment. This article must therefore be hereby marked ‘‘advertisement’’ in [email protected]. accordance with 18 U.S.C. §1734 solely to indicate this fact. †This paper is no. 159 in the series ‘‘Defense Mechanisms of Arthro- PNAS is available online at www.pnas.org. pods.’’ Paper no. 158 is ref. 22. 5570 Downloaded by guest on September 27, 2021 Ecology: Gonza´lez et al. Proc. Natl. Acad. Sci. USA 96 (1999) 5571 PA-injected virgin laboratory-raised females and their controls. Oral Sensitivity of L. ceratiola to PA. To obtain a measure These females were raised on the (2) diet and injected either of this parameter, individual female L. ceratiola were each with a PA solution (150 mg of monocrotaline in 5 ml of saline) given a live mealworm (presented in the same fashion as the or with solvent only (5 ml saline). Injection was effected with Utetheisa), which they promptly attacked. Once they initiated a microsyringe to one side of the posterior half of the abdomen feeding, they were stimulated by topical application of a on day 4 after pupal emergence. droplet of PA solution (100 mg of monocrotaline in 5 mlofegg Orb-Weaving Spiders (N. clavipes). Tests with this spider albumen), delivered with a micropipette on the mealworm as were carried out at Highlands Hammock State Park, Sebring, closely as possible to the spider’s chelicers. Control spiders Highlands County, FL, a preserved site densely shaded by live were stimulated with 5 ml of albumen alone. oak and cabbage palm, where N. clavipes occur naturally. The PA Analyses of Utetheisa Body Components. Samples were m feeding behavior of this spider is known (12). Typically, when weighed (wet weight) and extracted for 24 h with 500 lof y an insect flies into the spider’s web, the spider darts toward it buffer solution (2.7 g of monopotassium phosphate 2mlof y and bites it with the fangs to inject venom; the spider then triethylamine 0.4 ml of trifluoroacetic acid in 4 liters of water; envelopes the insect lightly in silk and carries it to the hub of pH adjusted to 3.0 with phosphoric acid). The samples then the web to eat it. Consumption is by suctional uptake of fluid were centrifuged, and the residue was reextracted with addi- m from the triturated, extraorally digested prey, a process that tional buffer solution (200 l). The two extracts were pooled, may take 1–3 h with insects the size of Utetheisa. and the mixture was analyzed by HPLC with a Hewlett– Packard 1090 Series II instrument with a diode array detector Presentation of Utetheisa to N. clavipes involved flipping l 5 3 m individual moths from vials into webs and monitoring the ( 205; C-18 BDS Hypersil column; 250 4.6 mm; 5- l particle size). The column was eluted (1 mlymin) with a buffer course of events. We knew from previous studies that N. y y clavipes reject wild Utetheisa and that they are sensitive to PA; solution acetonitrile mixture (92:8, vol vol). The PA ridelline they tend to consume mealworms (larvae of Tenebrio molitor) served as internal standard. PA Analyses of Thoracic Froth. only partially if these are treated by topical addition of When adult Utetheisa are squeezed gently or sometimes even merely touched, they emit crystalline monocrotaline (2). This spider was therefore well two bubbling masses of froth from the anterolateral margins of suited for our tests. Except where otherwise indicated, indi- the thorax (Fig. 1A). They share this propensity, which is vidual N. clavipes were given only a single Utetheisa and not obviously defensive, with a number of other arctiid species reused in any test. (13). To obtain froth samples for PA analyses, individual Wolf spiders (L. ceratiola). This spider was abundant on the female Utetheisa were squeezed gently with forceps, and the grounds of the Archbold Biological Station on sandy fire lanes, ensuing froth was picked up in microcapillary tubes, extracted where we spotted them at night by using head lamps that with ethanol (100 ml), centrifuged, and then reextracted with reflected strongly off their eyes. buffer solution (100 ml). After evaporating the ethanol from For laboratory testing, spiders were taken in vials and the first extract (nitrogen stream), the two extracts were pooled transferred singly into cylindrical plastic containers (17-cm and analyzed by HPLC as above. diameter; 11-cm height; with a shallow bottom layer of sand), Statistics.